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A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 (‘Human’) from 2 (‘Cattle’) in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

The study of genetic diversity in malaria populations is expected to provide new insights for the deployment of control measures. Plasmodium falciparum diversity in Africa and Asia is thought to reflect endemicity. In comprehensive epidemiological surveys reported here the genetic and antigenic structure of P. falciparum in the Venezuelan Amazon were studied over a 2-year period. DNA polymorphisms in glutamate-rich protein (GLURP), merozoite-surface protein 1 (MSP1) and MSP2 genes, in a multicopy element (PfRRM), all showed low diversity, 1 predominant genotype, and virtually no multi-clonal infections. Moreover, linkage disequilibrium was seen between GLURP, MSP1 and MSP2. Specific antibody responses against MSP1 and MSP2 recombinant antigens reflected the low genetic diversity observed in the parasite population. This is unexpected in a mesoendemic area, and suggests that the low diversity here may not only relate to endemicity but to other influences such as a bottleneck effect. Linkage disequilibrium and a predominant genotype may imply that P. falciparum frequently propagates with an epidemic or clonal population structure in the Venezuelan Amazon.

In view of the recent discovery (Molecular Cell6, 861–871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology119, 343–348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine.

Chloroquine-resistance in Plasmodium falciparum is associated with polymorphisms in a locus on or near the cg2 gene on
chromosome 7, and in the pfmdr1 gene on chromosome 5. In this study we typed P. falciparum DNA from uncomplicated
malaria cases in The Gambia in 1990, 1995 and 1996 for size polymorphism in the omega repeat of cg2, for sequence
polymorphisms in pfmdr1 at codons 86 and 184, in dhfr at codon 108 and in the msp2 gene. Chloroquine sensitivity tests
were conducted in vitro. A significant but incomplete association was found between the presence of the cg2 Dd2-like
omega repeat size polymorphism and in vitro resistance, and between the tyr-86 allele of pfmdr1 and in vitro resistance.
Furthermore there was strong linkage disequilibrium between the pfmdr1 asn-86 allele and the cg2 not Dd2-like omega
repeat allele located on different chromosomes. In contrast, no linkage disequilibrium was found between these alleles and
either the dhfr ser-108 allele or the msp2 IC sequence polymorphism. No significant linkage was measured between pfmdr1
asn-86 and phe-184 although these loci are separated only by 296 base pairs. Our results suggest that genetic elements
linked to the cg2 and the pfmdr1 genes are important determinants of chloroquine resistance. It can be concluded that the
observed linkage disequilibrium is maintained epistatically through selection by chloroquine.

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites
resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper
we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and
its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used
in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease
in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite
isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for
inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL)
and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and
HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of
inoculum size in in vitro susceptibility testing.

This study examines polymorphisms in 2 genes (pfmdr1 and cg2), which have been associated with resistance to
chloroquine in Plasmodium falciparum, to determine their value as predictors of resistance status. Among field samples
from children in Zaria, northern Nigeria, the Tyr-86 polymorphism in pfmdr1 and Ala-281 and the Dd2κ repeat of cg2,
were significantly associated. In 8 samples classified resistant by the micro-in vitro test, or, where this failed, by in vivo
trial, 7 showed the cg2 Dd2 type κ repeat, and 6 of these had both the Ala-281 allele and the pfmdr1 Tyr-86 allele. In 26
chloroquine-sensitive samples, none had this combination of 3 polymorphisms (P = 0·00002). This indicates 75%
sensitivity and 100% specificity in detection of resistance and shows a positive predictive value for resistant infections of
100%. The negative predictive value, because of sensitivity less than 100%, would depend on the prevalence of resistance.
Where prevalence of resistance is approx. 21% as in Zaria, the negative predictive value would be 94%, while in Gabon,
with a prevalence of ca 73% it would be 60%. The use of (cg2: Ala-281, Dd2κ. pfmdr1: Tyr-86) genotype detection as
a predictive epidemiological tool to examine the distribution of chloroquine-resistance in parts of Africa is therefore
possible. The sensitivity of detection of resistant strains still requires improvement.

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA
is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and
sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P. falciparum multidrug resistance
gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Plasmodium falciparum resistance to chloroquine has been
described in many
parts of the world particularly in Africa
where malaria is endemic. High levels of chloroquine resistance in our
study
area, Lambarene–Gabon, has led to the use
of an alternative regimen for treatment and prevention of P. falciparum
infection. In this study, we examined the in vitro
chloroquine sensitivity of 15 isolates from this area and assessed the
prevalence
of a putative chloroquine resistance associated Pfmdr1 polymorphism
(Asn86Tyr) using a novel allele-specific polymerase
chain reaction (PCR).
Only 4 of the isolates examined were chloroquine sensitive. The allele-specific
PCR shows that all
15 isolates carried the variant (86Tyr)
codon. Eleven of these were resistant to chloroquine suggesting a 73% agreement
between chloroquine resistance phenotype and the point mutation. This molecular
marker was examined in a further 73 Gabonese isolates, where 58 (79·5%)
showed
86Tyr and 15 (20·5%) showed 86Asn. In all,
4 (4·5%)
of the 88 isolates assessed carry both mutant and
wild-type codons, suggesting mixed parasite populations. The incomplete
agreement
found between chloroquine resistance phenotype and Pfmdr1 (86Tyr)
polymorphism would support the view that other genetic factors as well
as Pfmdr1 may
be involved in chloroquine resistance. While our results suggest a high
prevalence of 86Tyr
polymorphism in Lambarene, the Asp1246Tyr
polymorphism (a point mutation which to date has only been associated with
South American
P. falciparum) seems to be absent in our study area.

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM)
were used to treat Gambian children with uncomplicated falciparum malaria
in a randomized drug trial. Blood samples were taken immediately before
treatment (day 0), and at day 7 and day 28 after treatment. Samples
from those parasitologically positive at day 7 following treatment
(‘early positives’) and those positive at day 28 but negative
at day 7 (‘late positives’) have been studied by PCR followed
by restriction enzyme digestion to determine the allelic status of the
pfmdr 1 locus at the codon-86 position (asparagine
or tyrosine), previously associated with resistance to CQ. A
significantly higher prevalence of the tyr-86 allele was observed
in samples taken immediately before treatment (day 0) in the early
positives group when compared with the late positives
group. This suggests the tyr-86 allele contributes to drug resistance
in the early positives group. This association remained
significant for both CQ and AM groups, implying a common genetic basis
of resistance. Predominance of the allele at day
7 is consistent with a strong selection in the first week following
treatment. In the late positives group, a significantly
higher prevalence of the tyr-86 allele was observed in the samples at
day 28 when compared with those at day 0, suggestive
of selection during the period day 7 to day 28. Differences were observed
in the extent of this selection in the CQ and AM
groups. The samples were genotyped at 3 unlinked polymorphic loci. These
analyses suggested that most parasites
observed at day 7 were probably recrudescences whereas most of those at
day 28 were reinfections.

The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Homologues of the mammalian multidrug resistance gene have been identified in isolates and clones of Plasmodium falciparum and designated pfmdr1 and pfmdr2. Mutations in pfmdr1 have been associated with chloroquine resistance but confirmation could not be obtained in a genetic cross. We have examined the copy number and expression of pfmdr1 and pfmdr2 in chloroquine-sensitive and -resistant P. falciparum and have found no relationship between the copy number of either gene and chloroquine resistance. However, a marked correlation was seen between levels of mRNA transcribed for each gene and chloroquine resistance. Two transcripts of pfmdr1 were detected, and in the asexual blood cycle an 8 kb transcript appeared first, followed by the appearance of a 7 kb species.

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